The replication and segregation of the genome (the cell cycle) and the increase in bio-mass of individual cells (cell growth) must be coordinated in all cells. But the mechanism(s) underlying this coordination are very poorly understood, particularly in mammalian cells. The goal of Project 1 is to deconvolve cell growth and the cell division cycle, determine the molecular basis for the coordination of these two processes, and determine how these two processes and their coordination are altered in cancer. The proposed measurement platform will be developed by the Manalis lab in close collaboration with the Amon and Kirschner labs and will consist of separate modules for: obtaining synchronous populations of new-born daughter cells, measuring single cell growth rate, fixing cells, sequential storage of cells with Icnown identity, staining these cell in parallel with probes against mRNAs, proteins, and protein phosphorylation sites, and high-resolution fluorescent Imaging of the stained cells. Our investigations will be focused on white blood cells that proliferate without adhesion in culture, as these cells can be easily manipulated within our measurement platform and are inherently tolerant of fluid flow and shear stress. Similarly, we will begin our investigations using common lines of cultured cells, but we are cognizant of the danger of being seriously misled by transformed cells. Once our technologies are established, we will progress to studying primary lymphocytes derived from mice as well as untransformed epithelial cells. Finally, we will begin to directly apply our technologies to cancer by characterizing the cell growth and the cell cycles of rare cells isolated from a human lymphoma in collaboration with the Nolan lab at Stanford. RELEVANCE